The present application includes a Sequence Listing filed herewith on a single (CD-R) compact disc, provided in duplicate. The Sequence Listing is presented in a single file named xe2x80x9camended sequence.txtxe2x80x9d, last modified Nov. 26, 2002 2:32:50 am, and having 2,312,956 bytes, the disclosure of which is incorporated herein by reference in its entirety.
The present invention relates to a novel myosin-like protein particularly expressed in human heart and muscle, isolated nucleic acids encoding the myosin-like protein, compounds and compositions derivable directly or indirectly therefrom, and diagnostic and therapeutic methods for using the same.
Myosins are ubiquitous proteins that act as intracellular engines, typically motoring along tracks of actin filaments within the cell to drive a variety of cellular processes including muscular contraction, cytokinesis, membrane trafficking and signal transduction. Baker et al., Curr. Opin. Cell Biol. 10: 80-86 (1998). Given the range of intracellular passengers and itineraries, some form of the myosin protein is found in virtually all eukaryotic cells.
The myosin gene superfamily has at least seventeen members, or classes, encoded by multiple genes.
Mammalian cells have the largest number of myosin genes, with the 28 identified myosin genes belonging to nine classes. Sellers, Biochim. Biophys. Acta 1496: 3-22 (2000). The genome of the yeast, Saccharomyces cerevisiae, contains just 5 myosin genes. Brown, Curr. Opin. Cell Biol. 9: 44-48 (1997). Between these extremes, the nematode Caenorhabditis elegans has 14 identified myosin genes. Baker et al., J. Mol. Biol. 172: 523-535 (1997). Myosins are also found in plant cells, with myosin genes in classes VIII, XI and XIII expressed exclusively in plants.
The structure of myosin always includes one or two heavy chains and several light chains.
The heavy chain is composed of three structurally and functionally different domains. The head domain, which is highly conserved across the myosin family, functions to generate force, and contains actin-binding and ATP-binding sites; the ATPase activity of the head domain is activated by actin binding. The neck region, which mediates association with the light chains, regulates the adjacent head domain. The tail domain regulates the specific activity of each myosin.
The light chains of myosin I and myosin V are calmodulin, which is a calcium-binding regulatory subunit in certain enzymes. Myosin II is also regulated by calcium-binding light chains, but not calmodulin.
Together, the disparate myosin heavy and light chains permit myosins to serve disparate cellular roles.
For example, in brush border microvilli, myosin I, one of the two most abundant forms of myosin, links the microfilament bundles to the plasma membrane.
Myosin II, the other of the two most abundant myosin classes, forms dimers in muscle cells that associate to form thick filaments, which are part of the contractile apparatus.
In addition to its well characterized role in contraction and force production in skeletal, cardiac, and smooth muscles, myosin II is required for cytokinesis, cell motility, cell polarity/chemotaxis, maintaining cell architecture and development in nonmuscle cells. Sellers, Biochim. Biophys. Acta 1496:3-22 (2000).
Thus, contractile bundles, which comprise both actin and myosin II filaments, are found in numerous cell types. In epithelial cells, these bundles are called the circumferential belt and can function structurally (e.g., as an internal brace helping to control cell shape), or for motility (e.g., in wound healing, contraction seals the gap in a sheet of cells).
Myosin II is an integral component to the cytoskeletal substructure, acting to stiffen cortical membranes. In cytokinesis, myosin II plays an essential role performing the motor function for the contractile ring which constricts to form the cleavage furrow.
Myosin IXs, identified in rat, human and C. elegans, are expressed in a wide variety of tissues and cell types and are believed to be involved in intracellular signaling pathways. Myosin IX acts as a negative regulator of Rho (a G-protein), suggesting that it may control the Rho signaling pathways involved in cytoskeleton reorganization and other cellular processes. Bahler et al., Biochim. Biophys. Acta 1496:52-59 (2000). However the precise cellular functions of the myosin IXs and their exact roles in the Rho signaling pathways have still to be determined.
Membrane-bound myosins of various classes, particularly myosin I and myosin V, have been implicated in movement of vesicles within the cell. Due to its co-localization with Golgi membrane, myosin I is thought to move membrane vesicles between membrane compartments in the cytoplasm. Myosin I also serves as a membrane-microfilament linkage in microvilli.
In yet other examples of myosin function, protein secretion in yeast is disrupted by mutation in the myosin V gene, suggesting an important role for myosin V. In vertebrate brain tissue, myosin V is concentrated in the Golgi stacks and at the tips of membrane processes extending from neuronal cells. Espreafico et al., J. Cell Biol. 119: 1541 (1992). This type of membrane association would be consistent with the effects of myosin V mutations in mice, which adversely affect synaptic transmission, resulting in seizures and eventually death. In cell migration, different myosins localize to different regions of the cell. Myosin I localizes to the leading edge of a crawling amoeba, possibly participating in the translocation phase, whereas myosin II is more concentrated at the tail, where it is involved in retraction of the cell body.
Given their ubiquity, and the wide variety of tasks driven by myosins, it is not surprising that myosin defects have been implicated in a wide variety of diseases.
For example, as noted above, myosin V mutations in mice adversely affect synaptic transmission, resulting in seizures and eventually death.
Myosin and myosin-like genes have also been implicated in a number of human diseases.
For example, myosin plays a role in hypertrophic cardiomyopathy. An autosomally dominant form of the disease is frequently caused by a missense point mutation in exon 13 of the cardiac myosin heavy chain gene on chromosome 14. Less often, an abnormal cardiac myosin heavy chain hybrid gene is present.
As another example, mutation in the gene encoding myosin VIIA is responsible for some forms of Usher syndrome. Weil et al., Nature 374:60-61 (1995). Usher Syndrome is characterized by hearing impairment associated with retinitis pigmentosa.
Three classes of myosins, VI, VII and XV have been associated with genetic deafness disorders in mammals. Hasson, Am. J. Hum. Genet. 61:801-5 (1997); Redowicz, J. Muscle Res. Cell Mot. 20:241-248 (1999). Mutations in these myosins result in abnormalities in the stereocilia in the sensory cells of the inner ear of mice.
Other myosin-like genes may also be needed for normal stereociliary function in the inner ear, and mutations in these additional myosin-like genes may thus underlie or contribute to various forms of congenital deafness. For example, nonsyndromic hereditary deafness (DFNA17), mapped to chromosome 22q12.2-q13.3 in 1997, has now been associated with mutation in MYH9, a nonmuscle-myosin heavy-chain gene, located within the linked region. Lalwani et al., Am. J. Hum. Genet. 67:1121-8 (2000).
Despite the long-standing interest in myosins and sequence similarity among classes and across species, not all myosin and myosin-like genes have yet been identified, even in species that are genetically well characterized.
For example, a novel myosin-like gene (MysPDZ) has recently been cloned from mouse bone marrow stromal cells. The protein encoded by this newly identified gene contains a PDZ domain but no actin-binding domain. Furusawa et al., Biochem. Biophys. Res. Comm. 270: 67-75 (2000). PDZ domains are implicated in modular proteinxe2x80x94protein interactions, also binding to specific C-terminal sequences of membrane proteins. Gee et al., Biochem. 39: 14638-46 (2000).
MysPDZ appears to localize in the cytoskeleton. Furusawa et al. show significant similarity between MysPDZ and a genomic clone from human chromosome 17; a human orthologue has been identified which contains an ATP/GTP-binding site. Nagase et al., DNA Res. 3: 321-329 (1996). Another PDZ-domain containing protein in humans has been shown to associate with actin filaments, possibly through binding to alpha-actinin. Bauer et al., Blood 96: 4236-45 (2000).
And in humans, genes for other new myosin-like proteins have recently been cloned. See, e.g., WO 00/26372, and U.S. Pat. No. 6,001,593, the disclosures of which is incorporated herein by reference in its entirety.
Given the importance of myosins in normal cellular processes and in disease, there is a need in the art for access to as yet undiscovered myosin and myosin-like genes.
Furthermore, diseases of the heart and vascular system are a significant cause of human morbidity and mortality. Increasingly, genetic factors are being found that contribute to predisposition, onset, and/or aggressiveness of most, if not all, of these diseases. There is a need for methods and apparatus that permit prediction, diagnosis and prognosis of diseases of the human heart.
These and other needs in the art are satisfied by the present invention, which provides compounds, compositions and methods based upon the identification and cloning of a novel human myosin-like protein, hGDMLP-1, which is expressed at high levels in human heart and also in skeletal muscle.
In a first aspect, the invention provides an isolated nucleic acid comprising: (i) the nucleotide sequence of SEQ ID NO:1, the full length cDNA sequence, (ii) the nucleotide sequence of SEQ ID NO:2, which presents the hGDMLP-1 cDNA open reading frame, (iii) a nucleotide sequence that is a degenerate variant of the nucleotide sequence of SEQ ID NO:2, (iv) a nucleotide sequence that encodes a polypeptide with the amino acid sequence of SEQ ID NO:3, which is the full length amino acid sequence of hGDMLP-1, (v) a nucleotide sequence that encodes a polypeptide with the amino acid sequence of SEQ ID NO:3 with conservative amino acid substitutions; (vi) a nucleotide sequence that encodes a polypeptide with the amino acid sequence of SEQ ID NO:3 with moderately conservative amino acid substitutions, or (vii) a nucleotide sequence that is the complement of the nucleotide sequence of any one of (i)-(vi).
As would be understood, the isolated nucleic acid that encodes hGDMLP-1 can be either genomic or transcript-derived.
The invention also provides an isolated nucleic acid comprising a nucleotide sequence that hybridizes under high stringency conditions to a probe, the sequence of which probe (i) consists of SEQ ID NO:2, (ii) encodes a polypeptide having the sequence of SEQ ID NO:3, (iii) encodes a polypeptide having the sequence of SEQ ID NO:3 with conservative amino acid substitutions, or (iv) is the complement of (i)-(iii), wherein the isolated nucleic acid is nonidentical in sequence to GenBank accession no. AA993492 and is less than 50 kb in length.
As further described below, GenBank accession no. AA993492 is an anonymous EST identical in sequence to portions of exons 15 and 16 of hGDMLP-1. By xe2x80x9cnonidentityxe2x80x9d is intended that the nucleic acid of the present invention have fewer, additional, or at least one nucleotide different from that of AA993492.
The invention further provides an isolated nucleic acid comprising a nucleotide sequence that hybridizes under moderate stringency conditions to a probe, the sequence of which probe (i) consists of SEQ ID NO:2, (ii) encodes a polypeptide having the sequence of SEQ ID NO:3, (iii) encodes a polypeptide having the sequence of SEQ ID NO:3 with conservative amino acid substitutions; or (iv) is the complement of (i)-(iii), wherein the isolated nucleic acid is nonidentical in sequence to GenBank accession no. AA993492 and is less than 50 kb in length.
In a related aspect, the invention provides an isolated nucleic acid comprising a nucleotide sequence that encodes at least 8 contiguous amino acids of SEQ ID NO:3, wherein the isolated nucleic acid is nonidentical in sequence to GenBank accession no. AA993492 and is less than 50 kb in length.
The invention also provides an isolated polynucleotide comprising a fragment of at least 17 nucleotides of the isolated nucleic acid of any one of claims 1-3, wherein the isolated nucleic acid is nonidentical in sequence to GenBank accession no. AA993492 and is less than 50 kb in length.
In useful embodiments of the isolated nucleic acids of the present invention, the isolated nucleic acid encodes a polypeptide having ATPase activity. In further useful embodiments, the isolated nucleic acids encode a polypeptide capable of binding calmodulin. Typically, the nucleic acids of the present invention are expressed in both skeletal muscle and heart muscle.
In another aspect, the invention provides the isolated nucleic acid molecule of the present invention operably linked to one or more expression control elements.
The invention further provides a replicable vector comprising an isolated nucleic acid molecule of the present invention.
In a further aspect, the invention provides a host cell transformed to contain the nucleic acid molecule of the present invention, or the progeny thereof.
In a related aspect, the invention provides a method for producing a polypeptide, the method comprising: culturing the transformed host cell of the present invention under conditions in which the protein encoded by the nucleic acid molecule is expressed.
Accordingly, the invention provides hGDMLP-1 polypeptides. In some embodiments, the polypeptide of the present invention is produced by the method described above.
In other embodiments, the invention provides an isolated polypeptide selected from the group consisting of: (a) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:3; (b) an isolated polypeptide comprising a fragment of at least 8 amino acids of SEQ ID NO: 3; (c) an isolated polypeptide according to (a) or (b) in which at least 95% of deviations from the sequence of (a) or (b) are conservative substitutions; and (d) an isolated polypeptide having at least 65% amino acid sequence identity to the isolated polypeptide of (a) or (b).
In another aspect, the invention provides an isolated antibody or antigen-binding fragment or derivative thereof, the binding of which can be competitively inhibited by a polypeptide of the present invention.
The invention also provides a method of identifying binding partners for a polypeptide according to the present invention, the method comprising: contacting the polypeptide to a potential binding partner; and determining if the potential binding partner binds to said polypeptide.
In another aspect, the invention provides a method of modulating the expression of a nucleic acid of the present invention, the method comprising: administering an effective amount of an agent which modulates the expression of the nucleic acid. The invention further provides a method of modulating at least one activity of a polypeptide according to the present invention, the method comprising: administering an effective amount of an agent which modulates at least one activity of a polypeptide according to the present invention.
In another aspect, the invention provides a transgenic non-human animal or transgenic plant modified to contain a nucleic acid molecule of the present invention and a transgenic non-human animal unable to express the orthologue of the polypeptide of the present invnetion.
In another aspect, the invention provides a method of diagnosing a disease caused by mutation in human hGDMLP-1, comprising: detecting the mutation in a sample of nucleic acids that derives from a subject suspected to have said disease.
In a further aspect, the invention provides a method of diagnosing or monitoring a disease caused by altered expression of human hGDMLP-1, comprising: determining the level of expression of human hGDMLP-1 in a sample of nucleic acids or proteins that derives from a subject suspected to have said disease, alterations from a normal level of expression providing diagnostic and/or monitoring information.
The invention further provides pharmaceutical compositions, including compositions separately comprising the nucleic acids, proteins, antibodies, antagonists and agonists of the present invention and a pharmaceutically acceptable excipient.
In a related aspect, the invention provides a method for treating or preventing a disorder associated with decreased expression or activity of human hGDMLP-1, the method comprising administering to a subject in need of such treatment an effective amount of the pharmaceutical composition comprising the nucleic acid, protein, or hGDMLP-1 agonist of the present invention.
Analogously, the invention further provides a method for treating or preventing a disorder associated with increased expression or activity of human hGDMLP-1, the method comprising administering to a subject in need of such treatment an effective amount of the pharmaceutical composition comprising an antibody or antagonist of the present invention.
The invention provides diagnostic compositions comprising, e.g., the nucleic acid, antibody, or polypeptide of the present invention, the nucleic acid being detectably labeled. In some embodiments, the diagnostic composition is suitable for in vivo administration.
In another embodiment, the invention provides the isolated nucleic acid molecule of the present invention attached to a substrate, and a microarray, at least one probe of which microarray is a nucleic acid according to the present invention.
The invention further provides a method for detecting a target nucleic acid in a sample, the target being a nucleic acid of the present invention, the method comprising: a) hybridizing the sample with a probe comprising at least 30 contiguous nucleotides of a sequence complementary to the target nucleic acid in the sample under hybridization conditions sufficient to permit detectable binding of said probe to said target, and b) detecting the presence or absence, and optionally the amount, of said binding.
The invention also provides a fusion protein, the fusion protein comprising a polypeptide of the present invention fused to a heterologous amino acid sequence. In certain particularly useful embodiments, the heterologous amino acid sequence is a detectable moiety, such as a fluorescent moiety. In other embodiments, the heterologous amino acid sequence is an Ig Fc region.
In yet another aspect, the invention provides a method of screening for agents that modulate the expression of human hGDMLP-1, the method comprising: contacting a cell or tissue sample believed to express human hGDMLP-1 with a chemical or biological agent, and then comparing the amount of human hGDMLP-1 expression with that of a control.